Various molecular techniques have been utilized to increase expression of heterologous genes in various host systems such as E. coli, Saccharomyces cerevisiae, and other expression systems. Among the techniques used to increase the heterologous protein expression level is to increase the transcriptional rate, to increase the translational rate, and to decrease the degradation rate of the expressed proteins. Due to several desirable features, S. cerevisiae has been used as a host to express various proteins of interest. For example, heterologous proteins in S. cerevisiae are generally soluble. S. cerevisiae does perform glycosylation. Furthermore, there are well-established processes for large-scale production of yeast and yeast products. However, in general, the expression level in S. cerevisiae is low.
In general, S. cerevisiae has two types of expression plasmids, an autonomous replicating plasmid and an integrating plasmid. An autonomous replicating plasmid typically has the following salient features:
(1) A 2 .mu.circle DNA fragment for the autonomous maintenance of the plasmid. PA1 (2) A yeast selectable marker which can be nutritional and complements the host nutritional deficiency such as ura3, leu, his or dominant selectable markers such as the neomycin phosphotransferase gene. The expression of the neomycin phosphotransferase gene allows it to survive in the presence of the drug Geneticin.TM.. The promoter for the selectable marker genes is generally the native promoter of the gene itself (i.e., ura3, leu, his) or a promoter from another S. cerevisiae strain. PA1 (3) A strong S. cerevisiae promoter followed by multiple cloning sites for the insertion of genes of interest. PA1 (4) Procaryotic sequences such as an ampicillin resistance gene and colE1 origin of replication to permit growth and maintenance in E. coli. PA1 (1) A yeast selection marker which can be nutritional which complements the host's defect or one of the dominant selectable markers. PA1 (2) A strong S. cerevisiae promoter followed by multiple cloning sites for the insertion of genes of interest. PA1 (3) Procaryotic sequences such as an ampicillin resistance gene and colE1 origin of replication to permit growth and maintenance in E. coli.
The integration vector typically has the following salient features:
We have discovered that replacement of the promoter to the selectable marker with the weak promoter from the D-amino acid oxidase promoter from T. variabilis leads to the amplification of the plasmid containing the gene of interest.